Mechanisms of proteoglycan breakdown in connective tissue are complex and involve multiple agents and pathways. Aggrecan is the large aggregating chondroitin sulfate proteoglycan of cartilage. See, for example, Doege, et al. J. Biol. Chem. 266:894 (1991); GenBank/EMBL Accession Number M55172 (human aggrecan). In studies investigating the catabolism of aggrecan, experimental systems used have included monolayer cultures of primary chondrocytes from established chondrocyte cell lines (Hughes CE, et al., Biochem. J. (1995) 305:799-804; Lark M W et al., J. Biol. Chem. (1995) 270(6):2550-2556) and explant cultures using cartilage from a variety of anatomical sites and animal species (Flannery C R, et al. J. Biol. Chem. (1992) 267:1008-1014; Sandy J D, et al. Biochim Biophys Acta (1978) 543:536-44; Tyler J A, Biochem. J. 1985; 225:493-507). The addition of cytokines such as IL-1 and TNF have been extensively used as agents which promote the degradation of the extracellular matrix (Hughes C E et al., supra (1995) Morales T I, et al. Arch Biochem Biophys (1992) 293 (1):79-84; Fosang A J, et al. Matrix (1991) 11:17-24). In particular, these two cytokines have been shown to target the catabolism of aggrecan.
Several studies have now lead to a number of important discoveries which have defined specific cleavage sites along the protein core of aggrecan (Ilic M Z, et al. Arch Biochem Biophys (1992) 294 (1):115-22; Loulakis P, et al. Putative site(s) of enzymic cleavage. Biochem J (1992) 284:589-593; Sandy J D, et al. J. Biol. Chem. (1991) 266:8683-8685). In total there appear to be at least seven cleavage sites, and amino acid sequence analysis of cartilage proteoglycan breakdown products have defined two major sites of proteolytic cleavage in aggrecan which occur within the interglobular domain (IGD) between amino acid residues Asn.sup.341 -Phe.sup.342 ("AF") and Glu.sup.373 -Ala.sup.374 ("EA") (human sequence enumeration) . Doege K J, et al. J. Biol. Chem. (1991) 266:894-902. The AF cleavage site generates a C-terminal catabolic fragment (ending with the C-terminal sequence DIPEN) consisting of the 50-60 kDa G1 domain that remains in the tissue complexed to hyaluronate (Flannery C R, et al., 1992). In recent studies Fosang et al. (Trans. Orthop. Res. Soc. (1995) 20:4) have also identified N-terminal fragments (beginning with the N-terminal sequence FFG) from this cleavage site in synovial fluids from patients diagnosed with a variety of different arthritides (joint disease). Similarly, Witt et al (Trans. Orthop. Res. Soc. 1995; 20:122) have found these glycosaminoglycan-containing N-terminal fragments in media samples from control and IL-1 stimulated porcine explant cultures.
The EA cleavage site produces glycosaminoglycan-containing N-terminal fragments (ARG . . . ) that appear as the major aggrecan degradation products isolated from synovial fluid of patients with arthritis (Lohmander L S, et al. Arth. Rheum. (1993) 36:1214-1222). These N-terminal fragments are also found in media from cartilage explant cultures treated with IL-1 or retinoic acid (Hughes C E, et al., 1995; Sandy J D, et al., 1991). A recent study (Lark M W, et al., 1995) identified the C-terminal fragment (ending with the C-terminal sequence EGE) in rat chondrosarcoma cells treated with retinoic acid.
The proteolytic activity responsible for this Glu.sup.373 -Ala.sup.374 cleavage has not been identified but it appears to have specificity for Glu-Xaa peptide bonds where Xaa is Ala, Gly or Leu. This activity has been termed "aggrecanase." Fosang A J, et al. J. Biol. Chem. (1992) 267:19470-19474). As used in this specification, "aggrecanase" means a polypeptide (or polypeptides) that will catalyze cleavage of such Glu-Xaa peptide bonds in aggrecan. Although the sites of cleavage within the molecule have been well characterized, many of the agents responsible for generating the large number of different proteoglycan degradation products are still unidentified. It is believed that more than one enzyme is responsible for degradation of proteoglycans. Culture systems have been manipulated with a variety of agents that enhance proteoglycan catabolism, in an effort to discover the agent(s) responsible for its breakdown. However, these studies have been unsuccessful in defining any specific agent responsible for the degradation of aggrecan. Flannery C R, et al. Trans. Orthop. Res. Soc. (1993). Nonetheless, experimental data using purified aggrecan and modified aggrecan as a substrate for purified enzyme preparations has yielded some information on the specific cleavage sites of the enzymes involved in proteoglycan degradation. However, this in vitro work has not definitively ascertained the mechanism or identity of agents involved in the degradation of aggrecan in cartilage. Fosang A J, et al. (1992); Fosang A J, et al. J. Biol. Chem. (1991) 266:15579-15582; Fosang A J, et al., Biochem. J. (1994) 304:347-351.
In previous studies (Hughes C E, et al., supra, (1995); Hughes C E, et al. J. Biol. Chem. (1992) 267:16011-16014) a number of monoclonal antibodies have been developed that are specific for the products (proteoglycan aggregate catabolites) that have been cleaved by specific proteinases. These antibodies have proved useful as tools in studying the mechanisms of breakdown of proteoglycan in cartilage explant culture systems, by allowing monitoring of specific cleavage products.